The fluorescence of various chemical substances when subjected to light energy can provide useful information and has broad applications in a number of fields, including medical and dental imaging, microscopy, and non-destructive testing, for example. In fluorescence imaging, light of a first wavelength band is emitted and directed toward a sample that contains a fluorophore or other type of fluorescing agent. The fluorescing material may occur naturally; for example, teeth and bones are known to fluoresce when excited with light energy at various wavelengths. Alternately, one or more fluorescing agents may be introduced to the sample, such as by injection or coating, for example. The fluorescing substance emits light of a second wavelength band in response to the emitted excitation light. A sensing apparatus isolates the fluorescent light from the emitted excitation light according to wavelength. The amount and spectrum of light that is sensed provides an indicator of the composition of the sample material. In the optical system of the sensing apparatus, one or more optical filters are generally used for separating low-level fluorescent light of the second wavelength band from the higher-energy emitted light of the first wavelength band.
The schematic view of FIG. 1 shows a conventional optical apparatus 10 for fluorescence sensing. A light source 12 emits the excitation light of the first wavelength band toward a beamsplitter 20. Beamsplitter 20 directs this light toward a sample 30. Fluorescent light from sample 30 is then transmitted back through beamsplitter 20 and along a detection path to a detector 22. An excitation filter 24 blocks unwanted light from the excitation light path. An emission filter 26 blocks unwanted light from the detection path. Lenses 18 and other optical components are provided for conditioning the light path as needed.
Among difficulties with the conventional approach is the need for precision alignment of beamsplitter 20 and other components, with little tolerance for positioning error. As a further drawback, emission and detection light paths are orthogonal to each other, which makes it difficult to design optical apparatus 10 as a compact device. Because the fluorescent signal is much weaker than the emitted light energy, noise is a problem. Using high levels of excitation energy can add to this problem, since unwanted fluorescence can occur from other sources, such as from lenses and other components in the optical system itself. This type of “stray” fluorescence can be particularly difficult to suppress from the detection path. In light of these problems, apparatus of this type can require costly components and fabrication in order to reduce inherent noise and achieve needed performance levels.
Fluorescence sensing is even more challenging where fluorescence occurs at multiple wavelengths. The use of multiple fluorophores in a single sample, for example, has been shown to be of value in a number of applications. Glucose monitoring is one application in which multiple fluorophores can be used, wherein the ratio of fluorescence at two different wavelengths bands can be used to help monitor concentration levels in a sample, such as in a patient's skin tissue.
Thus, it can be seen that there is a need for a fluorescence sensing apparatus that is compact, that has reduced requirements for component alignment and cost, and that provides an improved signal-to-noise ratio for detecting fluorescent energy at multiple wavelengths.